Compounds and methods for scavenging dicarbonyl electrophiles

ABSTRACT

Compounds and methods of scavenging bifunctional electrophiles and reducing the occurrence of lysyl-levuglandin adducts in a subject in need thereof by administering a levuglandin adduct formation inhibiting amount of a compound of the following formula: 
     
       
         
         
             
             
         
       
     
     wherein the variables are defined herein.

PRIOR APPLICATIONS

This application claims benefit to U.S. Patent Application No.62/857,165, filed Jun. 4, 2019; and U.S. Patent Application No.62/978,183, filed Feb. 18, 2020. The contents of both applications areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under P30 ES000267, P50CA90949, R21CA201856, P01CA028842, P01CA116087, R01CA190612,R01DK053620, P30DK058404, T35DK007383 and P50 GM15431, awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

Embodiments of this invention relate to methods of inhibiting themodification of histones and DNA by levuglandins in a subject in needthereof by administering a compound of the present invention or apharmaceutically acceptable salt thereof.

Embodiments of this invention also are scavengers of bifunctionalelectrophiles, including bifunctional electrophiles that are generatedin vivo during carcinogenesis.

Embodiments of this invention also relate to inhibiting the formation oflevuglandin adducts.

Embodiments of this invention also relate to inhibiting the developmentof intramucosal carcinomas, as well as reducing the dysplasia that is acarcinoma precursor.

SUMMARY OF THE INVENTION

The burden of colorectal cancer (CRC) in the United States and worldwideis massive, representing the 3rd most common cancer and 2nd most causeof cancer deaths. The etiology of CRC is multifactorial and encompassesgenetic factors, environmental exposures, and/or inflammation. Despitemany decades of investigation, the molecular process by which healthycolonic epithelial cells transform remains largely undetermined. Inpatients with inflammatory bowel disease the risk for cancer isespecially high, leading to colitis-associated carcinoma (CAC). Thestrong mucosal immune response that occurs during colitis leads to thegeneration of effector molecules, including reactive oxygen species andprostaglandins, which are involved in the formation of dicarbonylelectrophiles, such as levuglandins or malondialdehyde. Theseelectrophiles are highly reactive with DNA and lysine residues, notablyin histones, thus favoring mutagenesis and somatic genomicabnormalities. However, the role of endogenous electrophiles incolorectal carcinogenesis remains unknown despite the fact that they canbe mutagenic. The present inventors have discovered that electrophilesare involved in colon carcinogenesis: 1) The electrophile proteinadducts on lysines are elevated in: i) human tissues from ulcerativecolitis (UC), UC dypsplasia, and CAC with a progressive increase; ii)colonic tumors of CRC patients; iii) dyplastic tumors of C57BL/6 micetreated with azoxymethane-dextran sulfate sodium (AOM-DSS), a model ofCAC, compared to non-tumor areas; iv) dysplastic tumors of mice withcolon-specific homozygous deletion of Apc; 2). The present inventorsshow that compounds of the present invention are potent scavengers thatreact with dicarbonyl electrophiles, and inhibit adduct formation. Onecompound of the present invention, 2-hydroxybenzylamine (2-HOBA) is anatural product that has successfully completed Phase I human testing atVanderbilt and has been shown to protect mice from oxidative damage inmodels of hypertension and Alzheimer's disease. Another compound of thepresent invention, 5-ethyl-2-hydroxybenzylamine (EtHOBA) is an analog ofHOBA, which can penetrate the nucleus and protect nuclear proteins fromelectrophilic oxidation. 3) In the AOM-DSS model, treatment of mice withEtHOBA significantly reduces adduct formation, tumor development anddysplasia, while enhancing antitumoral immune response. The presentinventors discovered that electrophiles have a key role in coloncarcinogenesis via genomic instability, epigenetic dysregulation, and/orsuppression of antitumoral immunity, and are key targets for cancerprevention.

Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has longbeen linked with the development of cancer, although little is knownabout any epigenetic effects of COX-2. A product of COX-2 activity,levuglandin (LG) quickly forms covalent bonds with nearby primaryamines, such as those in lysine, which leads to LG-protein adducts.Here, the present inventors demonstrate that COX-2 activity causesLG-histone adducts in cultured cells and liver tissue, detectablethrough LC/MS, with the highest incidence in histone H4. Adduction isblocked by a reactive dicarbonyl scavenger, which has no effect on COX-2activity as measured by PGE₂ production. Formation of LG-histone adductis associated with an increased histone solubility in NaCl, indicatingdestabilization of the nucleosome structure; this is also reversed withscavenger treatment. These data demonstrate that COX-2 activity cancause histone adduction and loosening of the nucleosome complex, whichcould lead to altered transcription and contribute to carcinogenesis.

Additionally, Helicobacter pylori (Hp) (H. pylori) induces an innateimmune response in epithelial and myeloid cells that leads toinflammation-associated cancer. The inflammatory effector molecules,such as prostaglandins and reactive oxygen species, can generate thebifunctional electrophiles: levuglandins (LG), malondialdehyde,4-oxo-nonenal, and acrolein. These molecules form covalent adducts onDNA bases and on lysines (Lys-LG) in histones, thus disruptingDNA/histone interactions and increasing risk for mutations. The presentinventors have discovered that compounds of the present inventionscavenge said electrophiles, which prevents adduct formation.

Accordingly, embodiments of the present invention include compounds andmethods for scavenging bifunctional electrophiles and/or LG-lysineadducts and/or lysyl-LG adducts in a patient in need thereof.

Another embodiment is a method of inhibiting formation of levuglandinadducts of histone and DNA in a subject in need thereof by administeringa compound of the present invention or a pharmaceutically acceptablesalt thereof.

Another embodiment of the present invention is a method of treatingpre-malignant lesions by administering a compound of the presentinvention or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a method of scavenginglevuglandins in nucleus of cells by administering a compound of thepresent invention or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is preventing malignantmutations of pre-cancerous conditions by administering a compound of thepresent invention or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is decreasing a subject'srisk of developing cancer by administering a compound of the presentinvention or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is the prevention ofcellular transformation to malignancy by administering a compound of thepresent invention or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a method preventingfurther mutations in colon, esophagus, breast, lung, pancreas,gastrointestinal cancer, and/or prostate cancers in a subject in needthereof by administering a compound of the present invention or apharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a method treating and/orpreventing a disorder resulting from elevated levels of LG-histoneadduct formation and adducts on DNA bases and on lysines (Lys-LG) inhistones.

Another embodiment of the present invention is when the disorderresulting from elevated levels of LG-histone adduct formation isneoplasia. In other embodiments, the neoplasia is a brain cancer, a bonecancer, an epithelial cell-derived neoplasia (epithelial carcinoma), abasal cell carcinoma, an adenocarcinoma, a gastrointestinal cancer, alip cancer, a mouth cancer, an esophageal cancer, a small bowel cancer,a stomach cancer, a colon cancer, a liver cancer, a bladder cancer, apancreas cancer, an ovary cancer, a cervical cancer, a lung cancer, abreast cancer, a skin cancer, a squamus cell cancer, a basal cellcancer, a prostate cancer, a renal cell carcinoma, a cancerous tumor, agrowth, a polyp, an adenomatous polyp, a familial adenomatous polyposisor a fibrosis resulting from radiation therapy.

Another embodiment of the present invention relates to treatment of adisease such as especially pre-malignant lesions of the gastrointestinaltract, colon or esophagus (Barrett's esophagus) or a colon cancer orother malignancies, preferably pre-malignant colon lesions or a coloncancer, in a subject in need thereof. The other malignancies to betreated according to the present invention are preferably selected fromthe group consisting of breast cancer, lung cancer, ovarian cancer,lymphoma, head and neck cancer and cancer of the esophagus, stomach,bladder, prostrate, uterus and cervix.

Another embodiment of the present invention is a method of inhibitingthe progression of a gastrointestinal cancer in a subject, comprisingadministering to the subject a levuglandin adduct formation inhibitingamount of a compound of the following formula:

wherein: R₁ is H, D, D₂, substituted or unsubstituted alkyl, substitutedor unsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; R₂ is H, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy,alkyl-alkoxy-R₄; R₃ is H, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, hydroxyl, nitro; R₄ H, D, D₂,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,carboxyl, substituted or unsubstituted aryl, or substituted orunsubstituted cycloalkyl; R₅ is H, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy; and pharmaceutical salts thereof.

In one aspect of this embodiment, the cancer is colorectal cancer.

In another aspect of this embodiment, the compound is2-hydroxybenzylamine, methyl-2-hydroxybenzylamine, orethyl-2-hydroxybenzylamine.

Another embodiment of the present invention is a method of mitigatingthe progression of pre-malignant lesions in a subject in need thereof byadministering an effective amount of a compound of the followingformula:

wherein: R₁ is H, D, D₂, substituted or unsubstituted alkyl, substitutedor unsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; R₂ is H, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy,alkyl-alkoxy-R₄; R₃ is H, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, hydroxyl, nitro; R₄ H, D, D₂,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,carboxyl, substituted or unsubstituted aryl, or substituted orunsubstituted cycloalkyl; R₅ is H, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy; and pharmaceutical salts thereof.

In another aspect of this embodiment, the lesions are in agastrointestinal tract.

Another embodiment of the present invention is a method of treatinggastrointestinal carcinoma in a subject in need thereof, comprisingadministering an H. pylori reducing effective amount of a compound ofthe following formula:

wherein: R₁ is H, D, D₂, substituted or unsubstituted alkyl, substitutedor unsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; R₂ is H, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy,alkyl-alkoxy-R₄; R₃ is H, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, hydroxyl, nitro; R₄ H, D, D₂,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,carboxyl, substituted or unsubstituted aryl, or substituted orunsubstituted cycloalkyl; R₅ is H, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy; and pharmaceutical salts thereof.

In aspects of this embodiment, the lesion exists in the colon,esophagus, breast, lung, pancreas, gastrointestinal tract, and/orprostate.

In other aspects, the inhibition step lowers levels of LG-histone adductformation in said subject.

In other aspects, the subject was first diagnosed with an H. pyloriinfection.

Another embodiment of the present invention is a commercial package orproduct comprising an adduction inhibitor, in particular those mentionedherein, or a pharmaceutically acceptable salt thereof, together withinstructions for the treatment of a disease such as especiallygastrointestinal cancer, pre-malignant gastrointestinal lesions, colonlesions or a colon cancer or other malignancies, preferablypre-malignant colon lesions or a colon cancer, in subject in needthereof.

According to the present invention, a patient is treated withtherapeutically effective amounts of an adduction inhibitor of thepresent invention, each according to a dosage regimen that isappropriate for the individual agent. For example, the adductioninhibitor may be administered once or more daily, on alternate days oron some other schedule—as is appropriate. One of skill in the art hasthe ability to determine appropriate pharmaceutically effective amountsof the combination components.

In the context of the present invention the terms “treatment” or “treat”refer to both prophylactic or preventative treatment as well as curativeor disease modifying treatment, including treatment of patients at riskof contracting the disease or suspected to have contracted the diseaseas well as patients who are ill or have been diagnosed as suffering froma disease or medical condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme and structure of the LG-lysyl adduct and thefragment ions monitored in positive ion mode (+H).

FIG. 2 shows immunohistochemistry for LG-protein adducts, human colonTMA. Scale bar, 50 μm.

FIGS. 3A and 3B show data from C57BL/6 mice were treated or not withvarious concentrations of EtHOBA. (A) Body weight was assessed everyday. (B) After 6 days, mice were euthanized and EtHOBA was measured inthe colon. n=5 mice/group.

FIG. 4 shows immunohistochemistry for LG-protein adducts. Scale bar, 50μm.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, and 5G show mice(n=10-12/group)±AOM-DSS±1.5 mg/ml EtHOBA. (A) Body weight; ″P<0.05compared to AOM-DSS-treated mice. (B) Tumor number. (C) Average tumorsize per mouse. (D) Tumor burden. (E) Number of adenomas. (F) Frequencyof diagnoses; ND, no dysplasia. (G) H&E staining; each arrow depicts atumor. Scale bar, 50 μm.

FIG. 6 shows representative data of levels of lysyl-L G adducts in thecolon of CRC patients. Adducts isolated from tumor (T) or non-tumor (NT)biopsies were analyzed by LC/EST/MS/MS.

FIGS. 7A, 7B, and 7C show data related to CDX2P-CreER^(T2);Apc^(fl/fl)mice (n=5) treated with 25 mg/kg tamoxifen. At day 33, animals wereeuthanized. (A) Colon, from proximal (left) to rectum (right), showingtumors (arrows). (B) H&E staining; each arrow depicts a tumor. Lowerpanel, high power view showing high grade dysplasia. (C) IHC for LG-protein adducts. Scale bar, 50 μm.

FIG. 8 shows immune cell infiltration score in AOM-DSS-treatedmice±EtHOBA.

FIG. 9 shows gene expression in the colon tissues of mice treated or notwith AOM-DSS±EtHOBA.

FIG. 10 shows a representative IF for CD68+NOS2+ cells in tumors ofAOM-DSS-treated mice±EtHOBA (n=3); NOS2; CD68; DAPI. Scale bar, 50 μm.

FIGS. 11A, 11B, 11C, 11D, 11E, and 11F are a series of graphs showingthat LG-lysine adducts of histones are found in cells and tissue,dependent on COX-2 activity. RAW264.7 mouse macrophage (A) and A549human lung carcinoma (C) cells were stimulated to express COX-2, thengiven 20 μM arachidonic acid (AA) or vehicle. A subgroup of cells waspreincubated 45 min with 50 μM indomethacin. As a measure of COXactivity, PGE₂ was determined by GC/MS from cell media prior to lysis (Band D). Nuclei were isolated, and histones were extracted and digestedto individual amino acids prior to LC/EST/MS/MS analysis. *, p<0.05;***, p<0.001 by ANOVA followed by Tukey's post-test (n≥5). (E). Histoneswere extracted from nuclei of rat liver, and analyzed as above forLG-lactam adduct. COX-2 protein was analyzed by Western blotting andplotted against lactam adduct levels. Each point corresponds to 1 liver,and shown is the line of regression (r²=0.7237). Pearson r=0.8507;two-tailed p=0.0152. (F) LC-MS chromatograph of histones isolated from arat liver with relatively high COX-2 expression (COX-2 band intensity of117 arbitrary units).

FIGS. 12A, 12B, 12C, 12D show LG-lysyl adducts are predominantlydetected on histone H4. (A). A Ponceau stain of a sample A549 histoneextraction is shown, along with band identities. Histones were extractedfrom nuclei in 0.4N H₂SO₄, resolved on 4-12% SDS-PAGE gradient gel andtransferred to nitrocellulose. H3 and H2B tend to run together as oneband. (B). RAW264.7 or A549 cells were stimulated to express COX-2 andgiven 20 μM ¹⁴C-AA for 1 h. Cells were lysed, nuclei were isolated, andhistones extracted, concentrated, and resolved on SDS-PAGE prior totransferring to nitrocellulose and exposing to film. Shown is theCoomassie stain of the SDS-PAGE gel (left) and the result ofautoradiography (right). The present inventors have observed thatPonceau, Coomassie, and silver stains each preferentially detectdifferent histone or acid-soluble proteins. (C-D). RAW264.7 (C) or A549(D) cells were stimulated to express COX-2, and treated with 20 μM AAfor 1 h prior to histone extraction. 350-400 μg of total histone wasloaded onto 4-12% SDS-PAGE gel and transferred to nitrocellulose.Individual bands were excised horizontally and proteins digesteddirectly off the nitrocellulose by serial incubations with Pronase andaminopeptidase. The results were analyzed by LC/ESI/MS/MS, and thechromatographs of the H3/H2B and H4 bands shown against the LG-lysylinternal standard. The H2A chromatograph is shown as a representativenegative result; no co-migrating peaks were seen in any other bands.

FIGS. 13A, 13B, and 13C show that the scavenger EtSA blocks LG-lysyladduct formation in RAW264.7 and A549 histones, without affecting COX-2activity. (A). Scavengers were screened in RAW264.7 cells for theability to decrease LG adduct formation on histones. Scavengers usedwere glucosamine (GA), 3-methoxysalicylamine (3-MoSA),pentylpyridoxamine (PPM), and 5-ethylsalicylamine (EtSA). Cells werestimulated to express COX-2, pretreated 45 min. with 500 μM scavenger orvehicle (H₂O), and given 20 μM AA for 1 h before lysing and extractinghistones. Histone proteins were analyzed by LC/ESI/MS/MS for LG-lysyllactam adduct, n=2. (B) Stimulated A549 cells were pretreated with 30,300, or 1000 μM EtSA prior to 1 h with 20 μM AA, and histones analyzedfor LG-lysyl adduct. *, p<0.05 by one-way ANOVA followed by Dunnett'smultiple comparisons post-test (n=3-5). (C). A549 cells were stimulated,pretreated 45 min. with 1000 μM EtSA or H₂O vehicle, and given 20 μM AAfor 1 h. Media was analyzed by GC/MS for PGE₂ (n=3). There was no effecton PGE₂ production at lower doses of EtSA (data not shown).

FIG. 14. LG-lysyl adduct formation on histone H4 decreases DNA-histoneinteraction. A549 cells were stimulated and given DMSO vehicle (C lanes)or 20 μM AA for 1 h (A lanes). A subgroup of cells was treated with 500μM EtSA 45 min prior to adding AA (E lanes). Nuclei were extracted with0.6, 0.9, or 1.2 M NaCl buffer, and the supernatant evaluated by Westernblotting for histone H4. Shown is a representative Western blot (A) aswell as the pooled results of 4 experiments (B). Different exposuretimes may have been used for the 0.9 M and 1.2 M bands. ***, p<0.001 byone-way ANOVA followed by Tukey's multiple comparisons post-test. NS,not significant.

FIGS. 15A, 15B, and 15C are graphs showing C57BL/6 (A) or INS-GAS mice(B) were infected for 8 weeks with H. pylori PMSS1. Lysyl-LG adductswere measured in the gastric tissues by LC/ESI/MS/MS. *P<0.05. (C)Lysyl-LG adducts concentration was determined in AGS cells infected(plain bars) or not (open bars) with H. pylori PMSS1 for 24 h, in thepresence or absence of EtSA. *P<0.05 vs. uninfected cells. § P<0.05 vs.infected cells without EtSA.

FIGS. 16A, 16B, 16C, 16D, 16E, and 16F show INS-GAS mice (8 per group)were infected or not with H. pylori strain PMS for 8 weeks and thentreated with EtSA (7.5 mg/ml) one week after infection. (A) EtSAconcentration in gastric tissues; **P<0.01, ****P<0.0001 vs. micewithout EtSA. (B) Frequency of diagnoses. IMC, intramucosal carcinoma;LGD, low grade dysplasia; ND, no dysplasia. (C) Quantification of extentof dysplasia and cancer as a percentage of tissue sections. (D) H&Estaining of INS-GAS mouse stomach tissues, showing carcinoma in aninfected mouse; scale bar, 50 μm. In B and C, *P<0.05, **P<0.01 vs.infected mice.

FIG. 17 is a graph showing reduction in the concentration oflysyl-levuglandin adducts in the stomachs of H. pylori infected micetreated with 2-hydroxybenzylamine for 8 weeks.

DESCRIPTION OF THE INVENTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are further disclosed and described, it is to beunderstood that they are not limited to specific synthetic methodsunless otherwise specified, or to particular reagents unless otherwisespecified, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular aspects only and is not intended to be limiting. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, examplemethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which need tobe independently confirmed.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “levuglandin scavenger” is a compound thatprevents reactive carbonyls such as levuglandin from reacting with DNAand proteins. Without being bound by theory or mechanism, this may occurby reacting with the carbonyls to form covalent adducts, thus preventingthem from forming adducts of DNA and proteins. Examples include covalentadducts on DNA bases and on lysines (Lys-LG) in histones.

One embodiment of the present invention is a method of treatinggastrointestinal cancer, including colorectal cancer (CRC). Despiteintensive efforts at colonoscopy-based screening, the disease burden ofCRC remains vast; it has the third highest incidence and the secondhighest mortality rate of all cancers. While there has been greatinterest in chemoprevention strategies, e.g. aspirin2, COX2 inhibitors,difluoromethylornithine+sulindac4, there is no effective strategy thathas impacted on clinical practice. Further, despite many studies ofoncogene and tumor suppressor gene alterations, the exact molecularmechanisms by which malignant lesions occur in the epithelium remainunclear. There are high-risk populations, including patients with: 1)longstanding inflammatory bowel disease (IBD), and 2) history ofhigh-risk adenomas or genetic abnormalities (e.g. familial adenomatouspolyposis coli; FAP) that are strong candidates for chemoprevention.

Under conditions such as inflammation and/or accumulated mutations,epithelial cells and adjacent immune cells express genes encodingenzymes involved in carcinogenesis, such as prostaglandin-endoperoxidesynthase (PGHS2; COX2), NADPH oxidases, and spermine oxidase. Theproducts of these enzymes (prostaglandins, O2D, and H2O2 plus3-aminopropanal) lead to formation of dicarbonyl electrophiles:levuglandins (LGs), malondialdehyde (MDA), 4-oxo-nonenal (4-O NE), andacrolein. Electrophiles may lead to immune dysfunction and neoplasticrisk by forming adducts with proteins and DNA11-15. However, the role ofthese reactive aldehydes in colon carcinogenesis is previously unknown.The present inventors have discovered that the level of LG-proteinadducts is increased in the colon tissues of i) human ulcerative colitis(UC), colitis-associated carcinogenesis (CAC), and CRC; ii) mice treatedwith azoxymethane-dextran sulfate sodium (AOM-DSS), a model of CAC; andiii) transgenic mice with a tamoxifen-inducible disruption of Apc usingthe colon-specific, caudal type homeobox 2 (CDX2) Cre(CDX2P-CreERT2;Apcff), which recapitulates sporadic/genetically-drivenCRC. The present inventors have also discovered that compounds of thepresent invention, including 2-hydroxybenzylamine (2-HOBA)(salicylamine) and 5-ethyl-2-hydroxybenzylamine (EtHOBA) react withdicarbonyl electrophiles at a rate 3 orders of magnitude faster thanlysine, thus preventing adduct formation with cellular macromolecules.2-HOBA is a natural product derived from buckwheat seeds. It is nottoxic or mutagenic and protects mice from oxidative damage in models ofhypertension and Alzheimer's disease. A phase I clinical safety trial inhumans has been completed at Vanderbilt University Medical Center (VUMC;NCT03176940)19, and other trials are ongoing. Additionally, the presentinventors show that compounds of the present invention significantlyreduce tumor development and dysplasia in the AOM-DSS model. The presentinventors also show that mice treated with compounds of the presentinvention show increased colonic immune responses to tumors.

Cyclooxygenase-2 (COX-2) expression is associated with the developmentof many cancers, and the enzyme plays a key role in the progression ofchronic gastrointestinal inflammation to cancer. Predictably, treatmentwith COX inhibitors decreases a person's total risk of cancer.Prevention studies as well as animal models suggest that increased COX-2activity is both an early event in carcinogenesis, which contributes tothe cellular transformation to malignancy, as well as a sustained eventin some colorectal and lung cancers that can be associated withmetastasis and poorer clinical prognosis. As predicted by these data,inhibiting COX-2 activity with non-steroidal anti-inflammatory drugs(NSAIDS) or COX-2-specific inhibitors over time reduces a person's totalrisk of colon, breast, lung, and prostate cancers.

Despite the promise of these drugs in cancer prevention, thegastrointestinal toxicity associated with long-term NSAID treatment andincreased cardiovascular events associated with COX-2-specificinhibitors limit their clinical use. A better understanding of thespecific downstream contributions of COX-2 to carcinogenesis could leadto new treatments that bypass these undesirable effects.

The product of COX-2, prostaglandin H₂(PGH₂), is converted enzymaticallyinto other prostaglandins, and indeed PGE₂ is a well-described promoterof carcinogenesis. However, depending on the animal model, deletion ofmicrosomal PGE₂ synthase-1 can either prevent or acceleratetumorigenesis, indicating that the contribution of COX-2 to cancer,particularly to cellular transformation, is probably multifaceted.

Besides enzymatic conversion, PGH₂ also spontaneously rearranges inaqueous solution to form the highly reactive levuglandins, LGE₂ andLGD₂. The levuglandins (LGs) constitute about 20% of total PGH₂rearrangement products. Newly formed LGs react almost immediately withfree amino groups, such as those in lysine, which leads to stablecovalent LG-protein adducts measureable by mass spectrometry (FIG. 1) orprotein-protein crosslinks. Following COX-2 activity, LG adducts ofprotein form in cells and in tissues. Proteins rich in lysine are thusespecially susceptible to adduction, and due to the perinuclearlocalization of COX-2 and a PGH₂ half-life measured in minutes, wespeculated that it would be possible for PGH₂ to cross the nuclearenvelope before rearranging to LGE₂, allowing formation of LG adduct onthe lysine-rich histones.

The present inventors have discovered LG-histone adducts in multiplecancer cell lines as well as rat liver, with highest measurable amountsof adduct on the H4 histone. Adduct formation is dependent on COX-2expression or activity. Lysines, with their short sidechain and positivecharge, are critical for histone ionic interaction with DNA, and theinventors find that this interaction is decreased with the introductionof a hydrophobic negative charge from LG. Covalent histone modificationsare a major method of controlling gene expression. Changes to lysylmodifications of histones are associated with human cancer. Thesefindings link COX-2 induction with perturbation of normal DNA-histoneinteractions and provide a novel role for the enzyme in carcinogenesis.

Importantly, the inventors have found a small-molecule salicylaminederivative that scavenges LG to reduce adduct formation on histoneswithout affecting COX-2 activity. In comparison with other scavengers,salicylamine, pyridoxamine and their other analogues, ethylsalicylaminewas most potent in protecting histones from modification bylevuglandins, indicating that it has the key property of beingtransported into the nucleus.

As stated above, Helicobacter pylori (Hp) induces an innate immuneresponse in epithelial and myeloid cells that leads toinflammation-associated cancer. The inflammatory effector molecules,such as prostaglandins and reactive oxygen species, can generate thebifunctional electrophiles: levuglandins (LG), malondialdehyde,4-oxo-nonenal, and acrolein. These molecules form covalent adducts onDNA bases and on lysines (Lys-LG) in histones, thus disruptingDNA/histone interactions and increasing risk for mutations. Embodimentsof the present invention scavenge said electrophiles, including5-ethylsalicylamine (EtSA), for example, which prevents adductformation.

As discussed further in the Examples, the human gastric epithelial cellline AGS was infected with Hp PMSS1±10 mM EtSA. FVB/N insulin-gastrin(INS-GAS) mice and Mongolian gerbils were infected for 8 wk with HpPMSS1 and 7.13, respectively; EtSA (0.75% in water) was given to animalsbeginning one wk post-infection. Lys-LG adducts, a marker ofelectrophile reactions, and EtSA were quantified by LC/EST/MS/MS.Histology was analyzed on H&E staining. DNA damage was evaluated bypH2AX staining. Hp colonization was quantified by culture. Mucosalimmune responses were determined by RNA profiling and by Luminex assay.Mice were imaged using PET/CT with ¹⁸F-FDG (inflammation marker) and¹⁸F—NaF (tumor marker).

Lys-LG adduct levels were increased in AGS cells infected with Hp andinhibited by EtSA. Similarly, Lys-LG adducts were significantlyincreased by 2.3-fold in infected mice compared to uninfected animals(p<0.05), and were completely inhibited by EtSA, which was found to bebioavailable in the stomach. There were decreases in frequency ofintramucosal carcinoma (from 69% to 28%; p<0.05) and extent of dysplasiaand carcinoma (p<0.05) in Hp-infected INS-GAS mice treated with EtSA.DNA damage was increased by 5.8-fold in infected mice and was decreasedby 71% with EtSA treatment (p<0.05). Levels of Hp colonization,histologic gastritis, and tissue cytokines and chemokines were notaffected by EtSA in INS-GAS mice. These data were confirmed by imaging:gastric uptake of the tumor marker ¹⁸F—NaF was induced by Hp anddecreased by EtSA, while uptake of the inflammation marker ¹⁸F-FDG wasincreased in infected mice, but not modified by EtSA. Similarly, thefrequency of gastric cancer development was reduced in gerbils treatedwith EtSA.

This shows that Hp-induced gastric carcinogenesis is inhibited by theelectrophile scavenger EtSA, without affecting inflammation. Thus,without being bound by theory or mechanism, embodiments of the presentinvention act downstream of inflammatory reactions by removingelectrophiles, which are a link between inflammation and the molecularalterations that lead to neoplastic transformation. The electrophilescavengers of the present invention serve as valuable chemopreventiveagents.

Compounds

It is understood that the following disclosed compounds can be employedin the disclosed methods of using or treating.

Examples of these compounds include, but are not limited to, compoundsselected from the formula:

wherein:R₁ is H, D, D₂, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl;R₂ is H, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, alkyl-alkoxy-R₄;R₃ is H, halogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, hydroxyl, nitro;R₄ H, D, D₂, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl;R₅ is H, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy; or analogs thereof; and pharmaceutical saltsthereof.

Without being bound by theory or mechanism, certain R₄ substituents arebelieved to impede metabolism by monoamine oxidase.

Examples of these compounds also include, compounds selected from theformula:

wherein:R₁ is CH₃ or alkyl;R₂ is H, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, alkyl-alkoxy-R₄;R₃ is H, halogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, hydroxyl, nitro;R₄ is a CH3 or alkyl;R₅ is H, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy;or analogs thereof; and pharmaceutical salts thereof.

In other embodiments, R₂ is H, R₅ is H, and R₄ is CH₃.

Further examples of compounds or analogs of the present inventioninclude compounds of the following formula:

wherein R₁, R₄, and R₆ are defined above. R_(4A) is independent from R₄and is H, D, substituted or unsubstituted alkyl, substituted orunsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; or an analog thereof, andpharmaceutical salts thereof.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl,sulfo-oxo, or thiol as described herein.

The term “alkoxy” group includes an alkyl group as defined above joinedto an oxygen atom having preferably from 1 to 10 carbon atoms in astraight or branched chain, such as, for example, methoxy, ethoxy,propoxy, isopropoxy (1-methylethoxy), butoxy, tert-butoxy(1,1-dimethylethoxy), and the like.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The terms “amine” or “amino” as used herein are represented by a formulaNA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen oroptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

Examples of compounds or analogs of the present invention includecompounds of the following formula:

or an analog thereof, and pharmaceutical salts thereof.

Further examples of compounds or analogs of the present invention are2-hydroxybenzylamine, methyl-2-hydroxybenzylamine, andethyl-2-hydroxybenzylamine.

Further examples of compounds or analogs of the present inventioninclude compounds of the following formula:

or an analog thereof, and pharmaceutical salts thereof.

Further examples of compounds or analogs of the present inventioninclude compounds of the following formula:

Further compounds or analogs may also be chosen from:

or an analog thereof.

The compounds may also be chosen from:

or an analog thereof.

The compounds of the present invention can also be chosen from:

The compounds of the present invention can be administered as the soleactive pharmaceutical agent, or can be used in combination with one ormore other agents useful for treating or preventing variouscomplications, such as, for example, lesions and cancer. Whenadministered as a combination, the therapeutic agents can be formulatedas separate compositions that are given at the same time or differenttimes, or the therapeutic agents can be given as a single composition.

The compounds of the present invention may be made up in a solid form(including granules, powders or suppositories) or in a liquid form(e.g., solutions, suspensions, or emulsions). They may be applied in avariety of solutions and may be subjected to conventional pharmaceuticaloperations such as sterilization and/or may contain conventionaladjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers, buffers etc.

For administration, the compounds of the present invention areordinarily combined with one or more adjuvants appropriate for theindicated route of administration. For example, they may be admixed withlactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they maybe dissolved in saline, water, polyethylene glycol, propylene glycol,carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanutoil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.Other adjuvants and modes of administration are well known in thepharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art.

In therapeutic applications, the compounds of the present invention maybe administered to a patient in an amount sufficient to reduce orinhibit the desired indication. Amounts effective for this use depend onfactors including, but not limited to, the route of administration, thestage and severity of the indication, the general state of health of themammal, and the judgment of the prescribing physician. The compounds ofthe present invention are safe and effective over a wide dosage range.However, it will be understood that the amounts of compound actuallyadministered will be determined by a physician, in the light of theabove relevant circumstances.

The compounds of the present invention may be administered by anysuitable route, including orally, parentally, by inhalation or rectallyin dosage unit formulations containing conventional pharmaceuticallyacceptable carriers, adjuvants, and vehicles, including liposomes. Theterm parenteral as used herein includes, subcutaneous, intravenous,intraarterial, intramuscular, intrastemal, intratendinous, intraspinal,intracranial, intrathoracic, infusion techniques, intracavity, orintraperitoneally. In a preferred embodiment, ethylsalicylamine isadministered orally or parentally.

Pharmaceutically acceptable acid addition salts of the compoundssuitable for use in methods of the invention include salts derived fromnontoxic inorganic acids such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydriodic, hydrofluoric, phosphorous, and thelike, as well as the salts derived from nontoxic organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. Such salts thus includesulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Also contemplated aresalts of amino acids such as arginate and the like and gluconate,galacturonate, n-methyl glutamine, etc. (see, e.g., Berge et al., J.Pharmaceutical Science, 66: 1-19 (1977).

The acid addition salts of the basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

EXAMPLES

The following examples and discussion are to be construed as beingexemplary of the present invention, and not intended to be limitingthereof.

Example 1. Increased Electrophile Adduct Formation in Patients with CAC

The present inventors performed immunohistochemistry (IHC) stainingusing the single chain antibody (Ab) D11, which specifically binds to LG-protein adducts, and a tissue microarray (TMA) from VUMC that containscolon tissues from patients with ulcerative colitis (UC), and associatedprecancerous and cancerous lesions. Histopathologic diagnoses wereestablished from clinical case material from surgical resections andconfirmed by H&E staining of the TMA. High levels of L G-protein adductswere present in active colitis, dysplasia, and carcinoma compared tonormal patients (FIG. 2). On the TMA, 7/9 (78%) high grade dysplasia(HGD) cores and 21/23 (91%) CAC cores were scored positive for L Gadducts. The strongest nuclear staining was observed in patients withCAC (FIG. 2), supporting the concept that electrophiles may thus affecthistones and DNA.

Example 2. Bioavailability of EtHOBA in the Colon

The present inventors analyzed the effect of various concentrations ofEtHOBA and its bioavailability in control mice. Animals that were given3.7 mg/ml EtHOBA lost weight on day 1, but did not lose additionalweight and did not die (FIG. 3A). Mice treated with 0.37-1.5 mg/ml hadno loss of body weight compared to untreated mice (FIG. 3A). After 6days of treatment, EtHOBA concentrations in the colon were analyzedusing liquid chromatography electrospray ionization tandem massspectrometry (LC/EST/MS/MS). The EtHOBA scavenger was readily detectedin mice treated with 1.5 and 3.7 mg/ml (FIG. 3B). There was i) nomacroscopic evidence of organ damage and ii) no change in colonweight/length in mice treated with 1.5 or 3.7 mg/ml EtHOBA (not shown).

Example 3. Compounds of the Present Invention Reduce the Development ofCAC

Animals receive an i.p. injection of the carcinogen AOM (12.5 mg/kg)followed by 3 cycles of 4% DSS, each for 4 days, over a total of 56-77days. To directly determine the role of electrophiles in coloncarcinogenesis, the present inventors tested the effect of EtHOBA inC57BL/6 mice in the AOM-DSS model. Mice were given 1.5 mg/ml EtHOBA oneday after AOM and continued throughout the experiments, except duringthe DSS cycles, to avoid mixing. The present inventors assessedelectrophiles in colons of mice treated with AOM-DSS, by IHC forelectrophile protein adducts. There was markedly increased staining inmice with experimental CAC compared to control animals, predominantly intumor areas (FIG. 4), including strong epithelial nuclear staining,indicating that electrophiles reach the nucleus and can affect histonesand DNA. There was a marked attenuation of L G-protein adducts in micetreated with the EtHOBA electrophile scavenger (FIG. 4). There was nochange in body weight in control mice treated for 56 days with EtHOBA(FIG. 5A), as with the acute treatment (FIG. 3A). At each cycle of DSS,mice had a decrease of body weight (FIG. 5A); there was less weight lossin animals given EtHOBA (FIG. 5A). Most importantly, there weresignificantly less tumors (FIGS. 5B, 5F), lower tumor size (FIG. 5C) andtotal tumor burden (FIG. 5D), and a reduced number of histologicadenomas (FIG. 5E) in the AOM-DSS group treated with EtHOBA. Histologicassessment further revealed that all the mice treated with AOM-DSS alonehad either high grade dysplasia (HGD), or low-grade dysplasia (L GD;FIGS. 5F-G). In total, without the scavenger treatment, 25% of mice hadHGD and the remaining 75% had L GD; in contrast, in those receivingEtHOBA, 20% had no dysplasia, none had HGD, and 80% had only L GD, asignificant overall difference in diagnoses (FIGS. 5F-G). These dataindicate that scavenging of electrophiles by EtHOBA can lead toreduction of inflammation-induced neoplastic transformation in thecolon.

Example 4. Increased Electrophile Adduct Formation in Patients withColon Cancer

The present inventors analyzed the abundance of lysyl-L G adducts byLC/ESI/MS/MS11,12 in tumor and non-tumor tissues (from our collaborator,Dr. K. Washington) from patients with sporadic CRC. The level of lysyl-LG adducts in tumors was higher than in normal tissues (FIG. 6),supporting the concept that electrophiles and their adducts aregenerated in the human colonic mucosa and are reactive in tumors. Thisalso demonstrates that lysyl-L G adducts can be measured from colontissues using LC-MS, which is needed for this project.

Example 5. Lysyl-LG Adducts are Increased in Mice with a Model ofEnetic/Sporadic Colon Cancer

To mimic human CRC associated with APC loss, the present inventors usedC57BL/6 CDX2P-CreER^(T2); Apc^(fl/fl) mice. APC loss is a sentinel eventin CRC, due to germline transmission as in FAP or its variants (e.g.attenuated FAP) with 100% penetrance. It is also a hallmark of sporadicCRC. This mouse model has been developed and optimized over the lastdecade. A major advance has been the use of the Apc double-floxed mice(Apc^(fl/fl)) mouse, which greatly increases the speed of tumorpenetrance compared to the single flox (Apc^(fl/+)), combined with thetamoxifen inducible Cre, which allows for precise control and timing ofthe model. A single dose of 25 mg/kg i.p. of tamoxifen for these mice.These mice lose Apc gene function in the colon when treated withtamoxifen and exhibit tumors. The present inventors have found that miceshow a 100% penetrance of tumors in male mice after tamoxifen, with manycolon and rectal tumors seen (FIG. 7A). Histologically, the adenomatoustumors are large and readily detected on Swiss-rolls, with features ofhigh-grade dysplasia (FIG. 7B). To verify the generation ofelectrophiles in this genetic/sporadic CRC model, the present inventorsstained the colon of the tamoxifen-induced CDX2P-CreER^(T2);Apc^(fl/fl)mice using the D11 antibody. Dysplastic glands displayed marked stainingfor L G protein adducts compared to surrounding areas (FIG. 7C) and tocontrol animals (not shown).

Example 6. An Electrophile Scavenger Increases Immune Cell Infiltrationin Mice with CAC

The present inventors used the tissues generated for FIG. 5 to studyimmune cell responses in AOM-DSS-treated mice±EtHOBA. It was discoveredthat more influx of immune cells in mice treated with EtHOBA compared tountreated mice (FIG. 8). EtHOBA had no effect on histology in controlmice (not shown). Together with the data depicted in FIG. 5, resultsindicate that the electrophile scavenger both reduces the development oftumors in the AOM-DSS model and stimulates immune cell response. Becausescavenging of electrophiles led to more immune cells in the colon, thissuggests that electrophiles dampen mucosal immune responses.

Example 7. The Colonic Antitumoral Immune Response is Increased byEtHOBA

Using the tissues from the same mice as in FIG. 5, the present inventorsanalyzed the expression of key genes encoding mediators of the innateand adaptive immune response. The transcripts of the prototype i) M1macrophage cytokines IL-1β (Il1b), TNF-α (Tnfa), IL-12A (Il12a), andIL-12B (Il12b), and the enzyme NOS2 (Nos2); ii) M2 macrophage enzymeARG1 (Arg1), iii) Th1 cytokine IFN-γ (lfng), and iv) Th17 cytokine IL-17(Il17) were each significantly induced in the non-tumor and tumortissues of AOM-DSS-treated mice compared to control animals (FIG. 11).Importantly, the overall pattern was that the genes encoding antitumoraleffectors were expressed at a higher level in mice that were givencompounds of the present invention. The following reached statisticalsignificance for an increase with EtHOBA treatment: Tnfa (tumor), Nos2(tumor), Il12a (non-tumor and tumor), Il12b (tumor), and Il17(non-tumor) in this experiment (FIG. 9), which was limited by thereduced number of mice in the EtHOBA group with tumors. In contrast, thegene encoding the immunosuppressive myeloid mediator AR G1 was notaffected by EtHOBA (FIG. 9). Using IF, we confirmed that mice treatedwith AOM-DSS mice that were given EtHOBA had increased CD68⁺NOS2⁺ (M1macrophages) in colon tumors (FIG. 10).

These results suggest that the treatment of mice with experimental CACwith an electrophile scavenger exhibit more innate immune responses,including in the tumors, and potentially enhanced adaptive immuneresponses. Because compounds of the present invention reduce the numberand progression of tumors in the colon (FIG. 5), one mechanism of actionmay be restoration of antitumoral immunity. The present inventorspreviously reported that mice with myeloid-specific deletion ofornithine decarboxylase, the rate-limiting enzyme for polyaminesynthesis, developed less tumors in the AOM-DSS model—a finding that wasstrongly associated with enhanced immune cell infiltration in thenon-tumor area and increased M1 cytokines and NOS233. These resultsindicated that polyamines impair antitumoral

Example 8. COX-2 Activity and Histone Adduction

Experimental Procedures

Materials

All reagents and chemicals were purchased from Sigma-Aldrich (St Louis,Mo.) unless otherwise noted. Methanol and acetonitrile were from FisherScientific (Pittsburgh, Pa.) and were HPLC grade or higher.[14C]-arachidonic acid (AA) was obtained from Perkin-Elmer Life Sciences(Boston, Mass.). The following reactive dicarbonyl scavenger moleculeswere synthesized by V. Amarnath as previously described:pentylpyridoxamine (PPM), 3-methoxysalicylamine (3-MoSA), and 5-ethylsalicyl amine (EtSA).

Treatment of Cells

To stimulate COX-2 expression, A549 or RAW264.7 cells were treatedovernight with 5 ng/mL IL-1B (A549) or 10 μg/mL LPS and 10 U/mL IFNγ(RAW264.7) in serum-free medium. When indicated, cells were pretreatedwith indomethacin, aldehyde scavengers (glucosamine, 3-MoSA, PPM orEtSA), or vehicle (ethanol for indomethacin, H₂O for scavengers) for 45min, and then given 20 μM arachidonic acid (AA) or DMSO vehicle for 1 hbefore lysing.

Histone Extraction

Cultured cells were lysed in hypotonic buffer (10 mM Tris/10 mM NaCl/3mM MgCl₂) containing 1 mM pyridoxamine and 100 μM indomethacin toprevent the artifactual formation of LGE₂ during the lytic process.After letting cells swell on ice, membranes were disrupted by additionof Triton X-100 (0.5% final concentration) and vortexing. Nuclei wereisolated by centrifugation for 10 min at 1000×g, and the resultingpellet washed with PBS. Histones were extracted in 0.4 N H₂SO₄,precipitated with trichloracetic acid, and washed with acetone. Withthis method, histones are the predominant proteins and contaminatingnuclear proteins are reduced (FIG. 3A). Histones were resolubilized indilute NaOH, and pH neutralized with HCl. Protein concentration wasdetermined using the method of Bradford. For tissue, a portion of frozenliver was homogenized in buffer containing 40 mM sodium citrate, and 1%Triton X-100 with 3 mM Trolox and 100 μM indomethacin. The supernatantwas separated from settled debris, and nuclei were pelleted at 500×g andwashed. Nuclear pellets were frequently transferred to fresh tubes toavoid contamination by floating lipid debris; any remaining was removedwith a cotton swab. Histones were extracted as above. All centrifugationsteps were carried out at 4° C.

Sample Preparation and Mass Spectrometry

Histone samples were prepared for mass spectrometry by addition ofammonium bicarbonate to 5 mM final concentration before digesting tosingle amino acids by protease step-digestion as previously described.In the case of immunoblots, proteins were digested directly off thenitrocellulose through incubation of the nitrocellulose strip in 30μg/mL Pronase in ammonium bicarbonate buffer, and later addition ofaminopeptidase. All samples were centrifuged at 2000×g for 10 min. afterfinal digestion to remove precipitate, spiked with 0.2 ng¹³C-lysyl-lactam internal standard, and purified on prepared tC18cartridges (Waters Corp., Milford, Mass.). Samples on tC18 cartridgeswere washed with water, then 30% methanol, before being eluted in 80%methanol and concentrated by evaporation. Samples were evaluated byelectrospray ionization (ESI) LC/MS/MS on a ThermoFisher TSQ Quantumtriple quadrapole mass spectrometer in positive ion mode and quantitatedby isotopic dilution as previously described, with the exception of areduced flow rate of 0.1 mL/min.

Measurement of PGE₂

A sample of cellular media was taken just prior to lysis and centrifugedto remove any cellular debris. For PGE₂ analysis, samples were spikedwith 2 ng of [²H₇] PGE₂ as an internal standard. Prostaglandins wereisolated and derivatized for analysis by GC/MS, operating in negativeion chemical ionization (NICI) mode and monitoring selected ions aspreviously described. For the [²H₄] PGE₂ internal standard, m/z=528. Toaccount for the deuterium-protium exchange at the position C12 of [²H₇]PGE₂, the summation of the signals obtained at m/z=530, m/z=531 andm/z=532 was performed.

Autoradiography

A549 or RAW264.7 cells were treated overnight to stimulate COX-2expression and given 20 μM ¹⁴C-AA (116 μCi) for 1 h. Histones wereisolated as described above and separated on 4-12% SDS-PAGE gels (LifeTechnologies), which were stained with Coomassie and exposed to film for2 weeks.

Salt Extraction

60-80% confluent A549 cells were stimulated and treated with AA±500 μMEtSA before scraping cells in lysis media for nuclear isolation asabove. After addition of Tritox X-100 and vortexing, 1.5 mL of eachsample was aliquoted into an eppendorf and centrifuged to separatenuclei. Pellets were washed with PBS and all buffer removed. Nuclearpellets were resuspended in extraction buffer containing 0.6M, 0.9M,1.2M or 1.5M NaCl (with 10 mM Tris, pH 7.5, 3 mM MgCl₂, 0.5% NP-40, andprotease inhibitor cocktail) and incubated 10 min on ice. Following thisextraction period, the nuclei were centrifuged at 16,000×g to obtain thesoluble fraction. This was sonicated, denatured at 95° C., and analyzedby SDS-PAGE and Western blotting, using Ponceau stain to visualizeproteins and confirm equal loading, and anti-H4 antibody (Abeam,Cambridge, Mass.).

Statistical Analyses

All data were analyzed using Prism software (GraphPad, La Jolla,Calif.). Data are expressed as means±SE, and statistical significancewas determined using one-way ANOVA followed by Tukey's post-test orDunnett's multiple comparisons post-test, when appropriate. A pvalue<0.05 was considered significant.

Results

Levuglandins Form Adducts on Histones in Cultured Cell and Whole Tissue

With mass spectrometry, the present inventors identified LG-lysyladducts on histones in RAW264.7 macrophages (FIG. 11A) as well as A549cultured lung epithelial cells (FIG. 11C). COX-2 is upregulated in thesecells upon cytokine stimulation, and addition of exogenous AA leads toformation of LG-histone adducts. Formation of these adducts is blockedwith indomethacin, further indicating a COX-dependent mechanism (FIGS.11A and 11C). Very few LG-histone adducts are formed in these cell lineswithout addition of exogenous AA, and PGE₂ analysis of cell media fromeach group indicates there is comparatively little endogenous AAmobilized following induction of COX-2 (FIGS. 11B and 11D). Although fewadducts are formed at basal levels in our cell lines, we find theLG-lysyl adduct in rat liver histones (FIG. 11E), where levels correlatewith COX-2 expression, demonstrating COX-2-dependent adduct formationunder physiological conditions.

LG-Histone Adducts are Restricted to Specific Histone Isoforms

Using 0.4N H₂SO₄ to extract histones results in a relatively purepreparation, with histones corresponding to known molecular weights(FIG. 12A). Incubation of ¹⁴C-AA with stimulated A549 or RAW264.7 cellsled to a ¹⁴C-containing band in the histone preparation thatcorresponded with H4 and H3/H2B, despite the fact that other histonesare represented in equal or greater quantity (FIG. 12B). The presentinventors treated stimulated RAW264.7 macrophages or A549 lung carcinomacells with 20 μM AA for 1 h and directly digested and analyzed theSDS-PAGE bands as labeled in FIG. 12A. The H4 band yielded a predominantpeak corresponding with the internal LG-lysyl standard, while lower orno signal was seen in the H3/H2B band and no corresponding peaks wereseen in other bands (FIGS. 12C and 12D, data not shown). Thus, there isconsistent evidence for formation of an LG-lysine lactam adduct on H4.The radiolabeled AA product adducted to H3/H2B probably includesstructures in addition to the LG-lysine lactam. These results, from twoseparate cell lines, suggest that there is specificity in the reactionof LG with histones.

The Scavenger 5-Ethylsalicylamine (EtSA) Reduces LG-Histone AdductFormation without Affecting COX-2 Activity

As demonstrated herein, in RAW264.7 cells, 5-ethylsalicylamine (EtSA)most effectively blocked adduct formation. Pentylpyridoxamine partiallyinhibited histone adduct formation, but was much less potent than5-ethylsalicylamine (FIG. 13A). EtSA also inhibited LG-histone adductformation in stimulated, AA-treated A549 cells, without affecting PGE₂production at the highest concentration tested (FIGS. 13B and C).

Formation of LG-Lysyl Adduct on Histone H4 Decreases DNA-HistoneInteraction

To examine the functional effect of LG-histone adduction, we performedsalt fractionation of A549 nuclei to determine histone solubility. Inthis assay, loosely-bound histone is released at lower saltconcentrations than tightly-bound proteins. The present inventorsdiscovered that in stimulated, AA-treated A549 cells, histone H4 waseluted at lower salt concentrations than in stimulated control cells;this was reversed after treatment with the scavenger EtSA (FIG. 14).

Discussion

As indicated herein, the present inventors established that COX-2catalysis can cause changes in DNA-histone interactions throughformation of LG-histone adducts, suggests a new hypothesis for thecontribution of COX-2 to the etiology of cancer. Oxidative damage isknown to cause N⁶-formylation of H1 histone, and epigenetic modificationaffecting COX-2 transcription is well-described, but the LG-lysylhistone adduct we describe here is an entirely novel finding that linksinflammation and COX-2 activation with histone modification.

The present inventors discovered COX-2 dependent formation of LG-histoneadducts in cells and tissues. Whereas COX-2 blockade by treatment withindomethacin decreases LG-histone adduct formation in A549 or RAW264.7cells, this method of antagonism cannot separate the myriad effects ofother COX-2 products from the effects of the LG-histone adducts. Thepresent inventors screened a number of small molecule levuglandinscavenger molecules for their ability to decrease LG-histone adduction.LG will react with these molecules three orders of magnitude faster thanlysine, and we have previously shown that scavenger treatment decreasestotal cellular levels of LG-lysine adducts without affecting PGE₂production. These scavengers are orally bioavailable, and able todecrease total LG-protein adduct levels when given to mice in drinkingwater. In future studies, aside from allowing investigation ofLG-protein modification independent of COX activity, use of thesescavengers may bypass the cardiovascular and gastric side effects seenwith COX inhibitors.

Interestingly, not all histones are targeted, but cellular LG adductsseem to preferentially form on the H4 and, to a lesser extent, onH3/H2B. Whether this specificity is a reflection of histone availabilityin the nucleosome, accessibility of lysine residues, or a more favorablemicroenvironment for adduct formation remains to be shown. Incubationwith [¹⁴C]-AA in cells led to a stronger autoradiographic band at H3/H2Bcompared to H4, while LC-MS analysis indicated that H4 was the majorform adducted by LGs. This discrepancy can be explained by severalmechanisms. Protein-associated radioactivity would come from any productderived from [¹⁴C] AA, including LGs but also PGJ₂/PGA₂ cyclopentenoneor arachidonate ester adducts. In addition, the occurrence of LG-lysyladducts is almost certainly underreported with our current approach. Ourinternal standard and mass spectroscopy method are specific fordetection of a single LG-lysyl adduct with an m/z equivalent to thelactam structure, but the initial Schiff base intermediate of LG-lysyladducts can oxidize to form other structure such as hydroxylactam orintraprotein or protein-DNA crosslinks, which would go undetected in ourmethod. For these reasons, the autoradiograms can not be quantitativelycompared with the LC/EST/MS/MS results as they do not measure the samemolecular structures.

H4, along with H2A, H2B, and H3 histones, comprise the histone octameraround which DNA is “packaged” into nucleosomes. The interaction ofhistone N-terminal tails with DNA is critical to DNA compaction andorganization, and is dependent on the numerous positively charged lysineand arginine residues present; a mesoscopic model demonstrates that H4tails are the most important in mediating internucleosomal interactions.A single lysyl acetylation on K16 of H4 modulates chromatin compactionand interaction of numerous chromatin-associated proteins; constitutiveacetylation of this residue confers a folding defect comparable todeletion of the entire H4 tail. After LG-histone adduct formation we dofind disruption of histone-DNA binding, resulting in increased DNAextraction in a salt solution. This decreased histone-DNA interactionmay increase DNA transcriptional access to previously silent oncogenes,and contribute to the development of cancer.

The complex patterns of lysyl acetylation and methylation comprise a“histone code” that regulates chromatin access and transcription; it isplausible that irreversible adduction of lysyl residues could disruptthis code, or directly alter the access of DNA-interacting proteins.Changes in histone modifications are known to result in altered DNAmethylation, deregulation of oncogenes, genomic instability, impairedDNA repair, and defects in cell cycle checkpoints. Changes in lysylmodifications of H4 in particular are a common hallmark of humancancers, and are associated with a global loss of DNA methylation.Further elucidation of the effects of LG-histone adduction on histonemodification, DNA-histone interactions, and transcription shouldincrease our understanding of the molecular mechanisms whereby COX-2contributes to cancer development and progression.

Example 9. Inhibition of Formation of Levuglandin Adducts in an In VivoAnimal Model in which Intramucosal Gastric Carcinoma Develops FollowingInfection with H. pylori

To demonstrate that bifunctional electrophiles are generated in vivoduring carcinogenesis, the present inventors analyzed the concentrationof lysyl-Levuglandin (lysyl-LG) adducts using liquid chromatographyelectrospray ionization tandem mass spectrometry (LC/EST/MS/MS), in thewhole gastric tissues of C57BL/6 mice (see FIG. 15A) and INS-GAS mice(FIG. 15B) infected by H. pylori. In both types of mice, lysyl-LG adductlevels were significantly increased in infected mice compared touninfected animals, demonstrating that electrophiles are locallygenerated during the infection. To further determine whetherethylsalicylamine (EtSA) prevents the formation of bifunctionalelectrophiles during H. pylori infection, the mice were treated or notwith EtSA 30 min prior to the infection. Our results show that lysyl-LGadduct concentration was significantly increased in H. pylori-infectedAGS cells when compared to uninfected cells and completely attenuated by10 μM EtSA (FIG. 15C).

The effect of a scavenger of levuglandins and other dicarbonyls wasinvestigated in transgenic INS-GAS mice, which have high circulatinggastrin levels and develop atrophic gastritis, and dysplasia andcarcinoma with H. pylori infection. Mice were infected with H. pylori,and then EtSA was administered in drinking water, starting 7 days afterinfection. After 8 weeks, EtSA levels were measured in the gastrictissues of uninfected and infected mice receiving this compound (FIG.16A). There were significant decreases in development of dysplasia (from37.5% to 14.3%) and carcinoma (from 37.5% to 0%) in H. pylori-infectedINS-GAS mice treated with EtSA (FIG. 16B). Extent of dysplasia andcarcinoma as a percentage of the gastric mucosa was also significantlyattenuated by EtSA (FIG. 16C). FIG. 16D depicts hematoxylin and eosin(H&E) staining of the gastric mucosa of infected INS-GAS mice showingintramucosal carcinoma in an infected mouse, characterized by irregularand angulated glands with infiltration of the lamina propria by tumorcells and desmoplastic stroma; in contrast, infected mice treated withEtSA showed low grade dysplasia characterized by irregular proliferationof glands that do not infiltrate the stroma. There was no dysplasia orcarcinoma in uninfected mice (FIG. 16D). It should be noted that gastriccolonization by H. pylori was not reduced by EtSA treatment (data notshown), demonstrating that the protective effect of EtSA was unlikelydue to an effect of EtSA on bacterial killing.

In summary, it is well established that in humans, H. pylori infectioninitiates an inflammatory process that leads to gastric cancer. Theresults of these investigations show that compounds of the presentinvention are useful for preventing the development of H. pylori-inducedgastric cancer in humans.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

We claim:
 1. A method of inhibiting the progression of agastrointestinal cancer in a subject, comprising administering to thesubject a levuglandin adduct formation inhibiting amount of a compoundof the following formula:

wherein: R₁ is H, D, D₂, substituted or unsubstituted alkyl, substitutedor unsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; R₂ is H, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy,alkyl-alkoxy-R₄; R₃ is H, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, hydroxyl, nitro; R₄ H, D, D₂,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,carboxyl, substituted or unsubstituted aryl, or substituted orunsubstituted cycloalkyl; R₅ is H, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy; and pharmaceutical salts thereof.2. The method of claim 1, wherein the cancer is colorectal cancer. 3.The method of claim 1, wherein R₁ is CH₃ or alkyl.
 4. The method ofclaim 1, wherein one of R₂, R₃, or R₅ is CH₃ or alkyl.
 5. The method ofclaim 1, wherein the compound is of the following formula:

and pharmaceutical salts thereof.
 6. The method of claim 1, wherein thecompound is 2-hydroxybenzylamine, methyl-2-hydroxybenzylamine, orethyl-2-hydroxybenzylamine.
 7. The method of claim 1, wherein thesubject is diagnosed with a H. pylori infection prior to theadministration step.
 8. A method of mitigating the progression ofpre-malignant lesions in a subject in need thereof by administering aneffective amount of a compound of the following formula:

wherein: R₁ is H, D, D₂, substituted or unsubstituted alkyl, substitutedor unsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; R₂ is H, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy,alkyl-alkoxy-R₄; R₃ is H, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, hydroxyl, nitro; R₄ H, D, D₂,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,carboxyl, substituted or unsubstituted aryl, or substituted orunsubstituted cycloalkyl; R₅ is H, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy; and pharmaceutical salts thereof.9. The method of claim 8, wherein the lesions are in a gastrointestinaltract.
 10. The method of claim 8, wherein R₁ is CH₃ or alkyl.
 11. Themethod of claim 8, wherein one of R₂, R₃, or R₅ is CH₃ or alkyl.
 12. Themethod of claim 8, wherein the compound is of the following formula:

and pharmaceutical salts thereof.
 13. The method of claim 8, wherein thecompound is 2-hydroxybenzylamine, methyl-2-hydroxybenzylamine, orethyl-2-hydroxybenzylamine.
 14. A method of treating or inhibiting theprogression of gastrointestinal carcinoma in a subject in need thereof,comprising administering an effective H. pylori infection reducingamount of a compound of the following formula:

wherein: R₁ is H, D, D₂, substituted or unsubstituted alkyl, substitutedor unsubstituted alkoxy, carboxyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted cycloalkyl; R₂ is H, substituted orunsubstituted alkyl, substituted or unsubstituted alkoxy,alkyl-alkoxy-R₄; R₃ is H, halogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy, hydroxyl, nitro; R₄ H, D, D₂,substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy,carboxyl, substituted or unsubstituted aryl, or substituted orunsubstituted cycloalkyl; R₅ is H, substituted or unsubstituted alkyl,substituted or unsubstituted alkoxy; and pharmaceutical salts thereof.15. The method of claim 14, wherein the compound is of the followingformula:

and pharmaceutical salts thereof.
 16. The method of claim 14, whereinthe compound is 2-hydroxybenzylamine, methyl-2-hydroxybenzylamine, orethyl-2-hydroxybenzylamine.
 17. The method of claim 14, furtherincluding the step of, prior to the administration step, diagnosing thesubject as being infected with H. pylori in the gastrointestinal tract.18. The method of claim 8, wherein the lesion exists in the colon,esophagus, breast, lung, pancreas, gastrointestinal tract, and/orprostate.
 19. The method of claim 1, wherein said inhibition lowerslevels of LG-histone adduct formation in said subject.